Our previous work revealed that CD44 hi CD62L lo memory-phenotype (MP) CD4+ T lymphocytes are spontaneously generated from naive precursors in steady state and exert innate-like effector function in pathogen infection (Kawabe T et al, 2017). In last years report, we further showed that MP cells consist of T-bet hi, T-bet lo, and T-bet- subpopulations and that differentiation of the former subset is homeostatically promoted by IL-12 produced by CD8 alpha+ type 1 conventional dendritic cells (cDC1). This tonic IL-12 production requires both basal priming via TLR-MyD88 signaling and CD40-CD40L augmentation. During the current report period we tested whether T-bet hi but not other MP cell subsets exert T cell receptor (TCR)-independent innate effector function. To do so we prepared CD4CreERT2 x TCR alpha flox mice to delete TCR on MP cells and crossed them to IFN-gamma-YFP T-bet-AmCyan double reporter animals. We found that T-bet hi but not T-bet- MP cells that are TCR- produce substantial levels of IFN-gamma in Toxoplasma infection, confirming the innate character of the same MP subset. We also sequenced the TCRs possessed by T-bet hi, T-bet lo, and T-bet- MP cells. While the TCR repertoire of T-bet- MP cells was diverse, that of T-bet hi cells was more limited, implying unidirectional differentiation from T-bet- to T-bet hi subsets in steady state. In additional experiments we asked whether foreign-derived agonists are necessary for both steady state IL-12 expression in cDC1 and T-bet hi MP differentiation or whether self-derived products are sufficient. We found that both germ -free (GF) mice which lack commensal bacteria and Ag-free (AF) mice that lack virtually all foreign Ags (including both commensal and food Ags) exhibited DC IL-12 levels and MP generation comparable to that seen in specific pathogen-free (SPF) mice. Thus, foreign-derived agonists appear to be dispensable for the generation of T-bet hi MP cells in steady state. Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions, principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through its inhibitory effects on cytokine and reactive oxygen species production. Recent in vivo experiments (Nair et al. J Exp Med 2018) have revealed that Irg-1-/-mice are unusually susceptible to Mtb infection displaying early mortality associated with increased bacterial loads, inflammation, and pathology. thus demonstrating an Irg1 regulatory axis that modulates inflammation to suppress Mtb-induced lung disease. During the report period we established a new project investigating the innate signals responsible for the induction of Irg1 in murine bone marrow derived macrophages by Mtb. Using macrophages from genetically deficient mice we established a partial requirement for TLR2/MyD88/NFkappaB signaling in the triggering of Irg1 mRNA by Mtb infection. Interestingly, the Irg1 response was strongly dependent on phagocytosis of the bacteria as well as the Mtb secreted virulence factor ESAT-6 and required Type I IFN but not IFN-gamma receptor signaling. In addition, Irg1 expression required the cytosolic DNA sensor STING. Based on these findings we hypothesize that Irg1 induction by Mtb is triggered by a two step process involving a TLR stimulus that is amplified by Type I IFN production triggered by bacterial phagocytosis leading to cytosolic activation of the STING pathway. The host factors that determine host susceptibility to Mycobacterium tuberculosis (Mtb) remain poorly defined. The microbiota has been identified as a key influence on the nutritional, metabolic, and immunological status of the host, although its role in the pathogenesis of Mtb is currently unclear. In studies performed last year, utilizing a rhesus macaque model of experimental Mtb infection we demonstrated that the microbiota composition at baseline could potentially predict the severity of TB disease progression. This year we were able to validate this observation in a separate cohort of age and gender matched animals as well as identify specific taxa associated with severe versus mild disease using shot-gun metagenomic sequencing. In an additional collaboration with Dan Barber's group we examined the role of the microbiota in the outcome of anti-PD-1 antibody treatment in Mtb infected rhesus macaques. We observed a significant correlation between microbiota composition and disease severity triggered as a result of PD-1 blockade. Together these non-human primate studies have revealed a potential interaction between each individual's microbiome and the outcome of MTb infection. Whether specific elements in the microbiota trigger direct effects on infection or serve as an indirect marker of other host susceptibility factors remains to be determined. Finally, we have started a new project that aims to understand the importance of the microbiome for the optimal function of alveolar macrophages, the first cells that encounter Mtb. Our initial experiments have demonstrated the alveolar macrophages (AM) from germ-free (GF) are more susceptible to in vitro Mtb infections in comparison to specific-pathogen free (SPF) mice. AMs from broad spectrum antibiotic treated mice display an intermediate phenotype. RNAseq analyses of sort purified AMs from SPF, GF and antibiotic treated mice reveal that the GF AMs possess a distinct transcriptional profile in comparison to the other groups thus suggesting a role for the microflora in the regulation of pulmonary immune cell function during tuberculosis.